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The role of Maf1 in age-associated phenotypes
Thesis   Open access

The role of Maf1 in age-associated phenotypes

Jessie M. Nagle
Master of Science (M.S.), Drexel University
Jun 2022
DOI:
https://doi.org/10.17918/00001116
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Abstract

RNA polymerases Cytology Molecular Biology
mTOR is a nutrient sensory kinase that regulates lifespan. mTOR controls cell growth, proliferation, and metabolism in response to nutrient availability. mTOR regulates the Maf1 protein by phosphorylation. When dephosphorylated and activated, Maf1 functions as a master inhibitor of RNA Polymerase III to repress transcription of tRNAs and 5S rRNAs. We hypothesize that the inactivation of Maf1 results in hyper-transcription, leading to transcription-associated DNA damage, senescence, and overall lifespan shortening. CRISPR/Cas9 genome-editing technology was utilized to establish a BJ-hTERT fibroblast cell line deleted of Maf1 to study the effect of Maf1 knockout on aging and senescence. We observed Maf1 depletion by shRNAs resulted in increased tRNA and 5s rRNA expression, as well as elevated expression of senescence markers such as p16 and p21. To investigate the role of Maf1 phosphorylation in aging and senescence, using a PCR site-directed mutagenesis approach, we have introduced mutations at phosphorylation sites on Maf1 to create unphosphorylated and phosphomimetic constructs. We have also introduced silent mutations to generate RNAi-resistant Maf1 expression vectors. These vectors will then be introduced into Maf1-shRNA expressing HCF cells to evaluate how Maf1 phosphorylation state affects age-associated phenotypes.

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